In order to identify and characterize thyroid stem/progenitor cells, we have taken several approaches. First, Side Population (SP) cells were obtained from mouse thyroid that demonstrated stem/progenitor-like properties. SP cells are characterized by their ability to efflux the vital dye Hoechst 33342 when analyzed by flow cytometry, due to expression of the ATP binding cassette (ABC)-dependent transporter ABCG2. SP cells, identified in various hematopoietic and non-hematopoietic adult tissues, are thought to be highly enriched for stem/progenitor cell activity. The population of thyroid SP cells was increased by partial thyroidectomy, a procedure that mimics acute thyroid injury, thereby revealing thyroid stem/progenitor cells as being required for active participation in the regeneration of injured thyroid. SP cells were subjected to microarray analysis as compared to non-SP cells with and without partial thyroidectomy, in order to find the genes that are specifically up or down-regulated in thyroid SP cells and/or after partial thyroidectomy. SP cells were further used to establish thyroid SP cell line that can be continuously cultured. These cultured SP cells have been subjected to a novel three-dimensional collagen gel culture system to determine whether they can reproduce functional thyrocytes. Histological analysis and laser capture microdissection in conjunction with microarray analysis were carried out to determine any differences in the histology and gene expression patterns before and after partial thyroidectomy in a whole thyroid. After partial thyroidectomy, immature-looking cells with clear or faintly eosinophilic properties, increased in the central part and the area near the cutting edge of the thyroid. Secondly, we focus our efforts on understanding whether NKX2-1, the homeodomain transcription factor that is critical for the genesis, homeostasis, and function of the thyroid, plays any role in maintenance and/or activity of thyroid stem/progenitor cells. This is based on our observation that the number of SP cells was reduced by approximately one-half in NKX2-1 heterozygous mouse thyroids as compared to wild-type mouse thyroids, and a recent study showing that NKX2-1 plays a role in thyroid carcinogenesis. We subjected NKX2-1 thyroid-specific conditional knockout mice, that hypomorphycally disrupts the NKX2-1 gene, to a thyroid chemical carcinogenesis study. The results revealed that NKX2-1 thyroid-specific conditional hypomorphic mice are more susceptible to a genotoxic chemical carcinogen than normal wild-type mice. These mice also more easily developed spontaneous adenomas. We also examined the role of p63 in development of the thyroid gland and Ultimobranchial Body (UBB), the origin of calcitonin-producing C cells, since the expression of p63, a member of the p53 tumor suppressor family, increases in NKX2-1 heterozygous mouse as compared to normal mouse. Further, p63 is thought to play a role in the commitment, maintenance, and differentiation of epithelial cells. For these studies, we used mice with various combinations of NKX2-1 and p63 wild-type, heterozygous, and null alleles. In the absence of p63, a normal thyroid gland developed, as revealed by expression of thyroglobulin and calcitonin, thus demonstrating that p63 is not required for thyroid development. However, in mice carrying the NKX2-1 null allele, the UBB remains as a cystic vesicular structure and/or in nested patterns consisting of p63-positive cells surrounding the vesicle and undifferentiated immature cells with occasional cilia lying inside. The cystic UBB was present even in the NKX2-1;p63 double-null mice. Although we do not know the exact nature of the undifferentiated UBB cells, they appear to be always associated with p63-positive cells. These results may suggest the possible stem/progenitor role for the undifferentiated UBB cells. The third approach we have taken is to do lineage-tracing experiments using thyroid-specific beta-galactosidase reporter mice that were subjected to partial thyroidectomy in conjunction with Bromo-deoxyuridine (BrdU) long label-retaining cells technique, which is considered to be the chosen method for demonstrating stem/progenitor cells. Beta-galactosidase is flanked by LoxP sites and is expressed upon activation of follicular cell specific thyroid peroxidase (TPO) gene that drives Cre recombinase expression. We found that mesenchymal cells expressing Sca1, but not beta-galactosidase, thyroid differentiation markers such as TPO and thyroglobulin, and those retaining BrdU expression, could be newly generated cells after partial thyroidectomy. Experiments are currently in progress.